Biochemical reactions require not only the presence of enzymes in the intracellular milieu but also essential small molecule cofactors (e.g., flavins and nicotinamides). Nicotinamide adenine dinucleotide (NAD+) is a common coenzyme that can serve as a conduit for electrons in oxidation/reduction reactions as well as a source of ADP in protein ADP-ribosylation. The synthesis of NAD+ can occur either through de novo synthesis or by a pyridine nucleotide salvage pathway. The de novo synthesis involves the conversion of L-aspartic acid into nicotinic acid mononucleotide (NAM) which is subsequently converted to nicotinic acid adenine dinucleotide, also known as deamino-NAD+ (NaAD). NAD synthetase (NadE) catalyzes the final reaction in the biosysthesis of NAD+. The substrate deamino-NAD+ combines with ATP to form the bound intermediate, NAD-adenylate. Ammonia is then added to the nicotinic acid carboxylate, releasing NAD+ and AMP (FIG. 1).
NAD synthetase belongs to a family of enzymes known as N-type ATP pyrophosphatases which share a common mechanism for adenylation of their substrates prior to amidation reactions. NAD synthetase also belongs to a class of enzymes known as amidotransferases which transfer ammonia to their substrates. Amidotransferases are typically characterized by the presence of two domains (present in a single polypeptide or as independent polypeptide subunits) one of which generates ammonia utilizing glutamine as a nitrogen source (glutamine amide transfer (GAT) domain), and the other which is responsible for the actual transfer of ammonia to the substrate. However the S. aureus NAD synthetase, like the Bacillus subtilis enzyme, does not contain a GAT domain and instead takes advantage of free ammonia as its nitrogen source. A separate GAT protein has yet to be identified in these organisms.
The gene encoding for the NAD synthetase has been shown to be essential for cell growth in several species including Bacillus subtilis, Escherichia coli, Salmonella typhimurium, and Rhodobacter capsulatus. Thus, identification of compounds that specifically inhibit the activity of bacterial NAD synthetase would have great therapeutic significance.